Effects of IFN-γ, TNF-α, IL-10 and TGF-ß on Neospora caninum infection in rat glial cells.

Neospora caninum causes abortion in cattle and neurological disorders in dogs. The immunological response to this parasite has been described as predominantly of the Th1 type. However, infected primary glial cell cultures release IL-10 and IL-6 but not IFN-γ. This suggests a rather protective response of the glia to avoid inflammatory damage of the nervous tissue. In this study, we investigated the effects of pro-inflammatory cytokines in primary mixed cultures of rat astrocytes and microglia infected with N. caninum. The cells were treated with either IFN-γ, TNF-α, anti-IL-10 or anti-TGF-ß antibodies and were infected with parasite tachyzoites 24h later. Trypan Blue exclusion and MTT assays were performed to test cell viability. It was observed that cytokines, antibody treatment and in vitro infection did not reveal significant cell death in the various culture conditions. Treatment with 50, 150 and 300 IU/mL of either IFN-γ or TNF-α reduced tachyzoites numbers in cultures by 36.7%, 54.8% and 63.8% for IFN-γ and by 27.6%, 38.4% and 29.7% for TNF-α, respectively. In the absence of IL-10 and TGF-ß, tachyzoite numbers were reduced by 52.8% and 41.5%, respectively. While IFN-γ (150 and 300 IU/mL) increased the nitrite levels in uninfected cells, parasite infection seemed to reduce the nitrite levels, and this reduction was more expressive in IFN-γ-infected cells, thereby suggesting an inhibitory effect on its production. However, TNF-α, IL-10 and TGF-ß did not affect the nitrite levels. Basal PGE(2) levels also increased by 17% and 25%; 78% and 13% in uninfected and infected cells treated with IFN-γ or anti-TGF-ß, respectively. Nevertheless, the antibody neutralization of IL-10 reduced PGE(2) release significantly. These results highlight the possibility of a combined effect between the IFN-γ and parasite evasion strategies and show that the IFN-γ, TNF-α, IL-10 and TGF-ß cytokines participate in parasite proliferation control mechanisms.

Toxocara canis infection is associated with eosinophilia and total IgE in blood donors from a large Brazilian centre.

OBJECTIVE: To investigate the association between Toxocara canis infection and total IgE levels and eosinophilia in blood donors from a large Brazilian city. METHODS: Two hundred and sixty-eight blood donors from a government blood bank were tested. No helminth infection was diagnosed by parasitological stool examination. Total IgE levels and T. canis infection status were determined by ELISA. Eosinophil levels were determined using an automatic blood cell counter. RESULTS: Toxocara canis IgG antibodies were found in 124 (46.3%); 102 (38.0%) had eosinophilia ≥4% and 29 (10.8%) had eosinophilia ≥10%, respectively; 140 (52.2%) individuals had total IgE antibodies above the cut-off levels. Both total IgE and eosinophil levels ≥10% were positively associated with the infection. CONCLUSION: This study revealed a high prevalence of T. canis infection in blood donors, highlighting the need for screening for this infection. It also demonstrated that this population otherwise healthy has higher levels of blood eosinophils and total IgE and that both parameters are associated with T. canis infection.

Antiproliferative, proapoptotic and morphogenic effects of the flavonoid rutin on human glioblastoma cells.

In this study, we investigated the effects of the flavonoid rutin (3,3',4',5,7-pentahydroxyflavone-3-rutinoside) on glioma cells, using the highly proliferative human cell line GL-15 as a model. We observed that rutin (50-100µM) reduced proliferation and viability of GL-15 cells, leading to decreased levels of ERK1/2 phosphorylation (P-ERK1/2) and accumulation of cells in the G2 phase of the cell cycle. On the other hand, 87.4% of GL-15 cells exposed to 100µM rutin entered apoptosis, as revealed by flow cytometry after AnnexinV/PI staining. Nuclear condensation and DNA fragmentation were also observed, further confirming that apoptosis had occurred. Moreover, the remaining cells that were treated with 50µM rutin presented a morphological pattern of astroglial differentiation in culture, characterised by a condensed cell body and thin processes with overexpression of GFAP. Because of its capacity to induce differentiation and apoptosis in cultured human glioblastoma cells, rutin could be considered as a potential candidate for malignant gliomas treatment.

Neospora caninum: early immune response of rat mixed glial cultures after tachyzoites infection.

Neospora caninum causes neurologic disease in dogs and abortion in cattle. Little is known about the immune response of the CNS against this protozoan. The aim of this study was to evaluate production of IL-6, IL-10, TNF-alpha, IFN-gamma, and NO in rat mixed glial cell cultures infected by N. caninum. IFN-gamma was not observed. The mean cytokine released after 24 and 72 h of infection were 3.8+/-0.6 and 3.7+/-0.6 pg TNF-alpha/mg protein and 2.7+/-0.69 and 4.1+/-0.64 pg IL-10/mg protein, respectively, and more than 8.0 pg IL-6/mg protein for both time points. NO levels increased 24h post-infection (2.3+/-0.8 pg/mg protein) until 72 h (4.2+/-1.1 pg/mg protein) and the number of tachyzoites reduced with the time. Our results show high levels of regulatory cytokines that may suppress the harmful effects of IFN-gamma; high levels of TNF-alpha and NO may represent an effective response by infected glial cells against N. caninum.

Evaluation of the humoral and cellular immune response to different antigens of Corynebacterium pseudotuberculosis in Canindé goats and their potential protection against caseous lymphadenitis.

Corynebacterium pseudotuberculosis is the etiologic agent of caseous lymphadenitis, a disease that affects goats and sheep, and can cause severe economic losses. In this study, four different antigenic extracts were obtained from the attenuated strain T1, which was isolated in the state of Bahia (Brazil). Forty-four Canindé breed goats were divided in five groups, each receiving a different antigen solution and saline buffer as a control. The humoral response was monitored through the identification of specific IgG by indirect ELISA and Western Blotting, and the production of IFN-gamma was followed in order to observe the activation of cellular response. After twelve weeks of antigen inoculation, the animals were challenged with 2 x 10(5)CFU of a wild strain, also isolated in Bahia, and necropsy was performed on all animals twelve weeks afterwards. It was observed that the attenuated bacteria gave a protection of 33.3%, in addition to the weak humoral response elicited. Animals inoculated with secreted antigen associated with Freund's incomplete adjuvant and oligodeoxynucleotide containing unmethylated CpG dinucleotides (CpG ODN) showed a strong humoral response, but this inoculation could not prevent the spread of challenge bacteria in the majority of animals. These results demonstrate the immunogenic potential of the attenuated T1 strain in the development of a vaccine against caseous lymphadenitis in goats.

The flavonoid rutin induces astrocyte and microglia activation and regulates TNF-alpha and NO release in primary glial cell cultures.

Astrocyte and microglia cells play an important role in the central nervous system (CNS). They react to various external aggressions by becoming reactive and releasing neurotrophic and/or neurotoxic factors. Rutin is a flavonoid found in many plants and has been shown to have some biological activities, but its direct effects on cells of the CNS have not been well studied. To investigate its potential effects on CNS glial cells, we used both astrocyte primary cultures and astrocyte/microglia mixed primary cell cultures derived from newborn rat cortical brain. The cultures were treated for 24 h with rutin (50 or 100 micromol/L) or vehicle (0.5% dimethyl sulfoxide). Mitochondrial function on glial cells was not evidenced by the MTT test. However, an increased lactate dehydrogenase activity was detected in the culture medium of both culture systems when treated with 100 micromol/L rutin, suggesting loss of cell membrane integrity. Astrocytes exposed to 50 micromol/L rutin became reactive as revealed by glial fibrillary acidic protein (GFAP) overexpression and showed a star-like phenotype revealed by Rosenfeld's staining. The number of activated microglia expressing OX-42 increased in the presence of rutin. A significant increase of nitric oxide (NO) was observed only in mixed cultures exposed to 100 micromol/L rutin. Enhanced TNFalpha release was observed in astrocyte primary cultures treated with 100 micromol/L rutin and in mixed primary cultures treated with 50 and 100 micromol/L, suggesting different sensitivity of both activated cell types. These results demonstrated that rutin affects astrocytes and microglial cells in culture and has the capacity to induce NO and TNFalpha production in these cells. Hence, the impact of these effects on neurons in vitro and in vivo needs to be studied.

Astroglial cells in primary culture: a valid model to study Neospora caninum infection in the CNS.

The protozoan Neospora caninum has a veterinary importance because it causes abortion in cattle and neuromuscular alterations in dogs. We infected rat astrocytes, in vitro, with different concentrations of N. caninum. Astrocytes responded to infection by producing the pro-inflammatory cytokine TNF-alpha and the neurotoxic-free radical NO, 24 and 72 h post-infection. These data suggest that astrocytes, which are essential for brain function, are targets for the parasite and this represents a practical and valid model to study the effects of N. caninum on the CNS.

Anti-inflammatory and anti-nociceptive activities of Pluchea quitoc (DC.) ethanolic extract.

Pluchea quitoc DC. (Asteraceae), a plant widely distributed throughout Brazil and popularly known as "quitoco", "madre-cravo" or "tabacarana", is used in traditional medicine for the treatment of inflammation, as well as of digestive and respiratory diseases. The anti-inflammatory and anti-nociceptive activities of the ethanolic extract (EE) from aerial parts of this plant were evaluated in mice and rats. Oral treatment with the EE (1-2g/kg, p.o.) decreased the paw oedema induced by carrageenan in rats, showed anti-nociceptive effects on the tail-flick test and on acid-induced writhing in mice, and inhibited both phases of pain (neurogenic and inflammatory) of the formalin test in rats. Topical application (EE 1.25, 2.5 and 5.0mg) inhibited the ear oedema induced by croton oil in mice. The results support the folkloric use of the plant in inflammatory processes.

Neospora caninum: infection induced IL-10 overexpression in rat astrocytes in vitro.

The effect of Neospora caninum, a parasite that causes abortion and neuromuscular changes, has been investigated on a major population of neural cells, the astrocytes. Highly enriched astroglial primary cultures obtained from neonatal rats were infected after 21 days of culture. Astroglial reactivity, IL-10 and IFN-gamma expression, and cell viability (lactate dehydrogenase activity, metabolization of tetrazolium salt, and trypan blue exclusion assay) have been investigated after 24 and 72 h of infection. Astroglial hypertrophy, gliofilament reorganization, metabolic changes suggesting hypoxia and a strong IL-10 release have been observed in the infected cells. These results show that neural cells are targets for the parasite and that astrocytes may contribute to the CNS immune response to the parasite.

Serologic immunoreactivity to Neospora caninum antigens in dogs determined by indirect immunofluorescence, western blotting and dot-ELISA.

Neospora caninum, is a coccidian protozoan known as a major cause of bovine abortion and canine neuropathies. The aim of the present study was to develop a reliable and quick test to detect antibodies to N. caninum in dog sera. Sixty-five serum samples from dogs, including 35 positive and 30 negative for N. caninum antibodies were used for standardization of the test. In parallel, immunoreactivity of the sera to Toxoplasma gondii antigens was investigated using a passive agglutination test. A dot-ELISA test, using soluble extract of N. caninum tachyzoites on nitrocellulose ester membranes, was developed and standardized. SDS-PAGE and complementary analysis of reactivity by Western blotting were used for the characterization of the immunoreactive fractions of all tested sera. The sensitivity and specificity of the dot-ELISA were 94 and 73%, respectively, compared to IFAT at a cut-off of 1:50, and 87 and 100% compared to IFAT at a cut-off of 1:25. Among the sera that tested positively for both IFAT and dot-ELISA, only 8.6% were reactive to T. gondii. The most immunoreactive fractions in Western blots were the 14-, 33-, 42- and 55 kDa bands, with percentages of 42, 60, 42 and 37%, respectively. The 60 kDa band showed a non-specific reaction in 43% of neosporosis-negative animals by both dot-ELISA and IFAT. These results indicate that the dot-ELISA using N. caninum antigen present good sensitivity and specificity, and might be used as a screening test to detect antibodies to N. caninum in dogs.

Phenotype analysis of lymphocytes of workers with chronic benzene poisoning.

Lifetime exposure to benzene is associated to a variety of blood disorders, and except for the risk of cancer, almost nothing is known concerning health impairment in individuals who are no longer exposed. In Brazil, this exposure is one of the serious problems in workplaces, and many workers have been laid off their jobs due to this intoxication, particularly in the State of Bahia, the largest producer of benzene in Latin America, which is the area of this study. From a larger study to describe health effects and genetic polymorphisms among workers with chronic benzene poisoning (CBP), this previous specific investigation analyzes the association between CBP and the pattern of sub-populations of lymphocytes. The study was performed with a CBP group (n=24) and a control group with other occupational diseases (n=24); both were selected at the Workers Health Study Center in the State of Bahia, Brazil. Clinical and epidemiologic variables were collected from medical records and from a detailed questionnaire. The average age was similar in the two groups (51.1 and 50.7, respectively). Analyzing the mean proportions of the sub-populations of lymphocytes, statistically significant differences were found for T cytotoxic cells (TCD8) (27.9; 19.4; p=0.002) and T helper memory cell (CD4CD45RO) (31.2; 37.0; p=0.015), respectively, for the CBP group and control group. These results should be viewed with caution because of the small sample size, but they strengthen a previous impression that workers exposed to benzene have their immune system impaired, even in the long term, which may contribute to some disorders and carcinogenesis process. These workers must be strictly followed up in a medical surveillance program. Although this problem has been known for a long time, this is the first attempt to study these specific effects in Brazil.

Three-phase partitioning as an efficient method for extraction/concentration of immunoreactive excreted-secreted proteins of Corynebacterium pseudotuberculosis.

The three-phase partitioning (TPP) technique was used upstream to isolate/concentrate secreted proteins from Corynebacterium pseudotuberculosis cultured in a complex liquid medium. Several parameters of the TPP technique (15, 30, or 60% ammonium sulfate concentration; 4.0, 5.5, or 7.0 pH; and primary (n) or tertiary (t)-butanol solvent isomer) were varied to determine the optimal recovery of serologically and cellularly immunoreactive extracted proteins. A TPP extraction made with 30% ammonium sulfate and an initial pH of 4.0 gave the best humoral and cellular immunoreactivity of caseous lymphadenitis infected goats. In particular, two immunogenic secreted (16 and 125 kDa) proteins, which had not been found by other extraction methods, were identified.

Experimental Corynebacterium pseudotuberculosis primary infection in goats: kinetics of IgG and interferon-gamma production, IgG avidity and antigen recognition by Western blotting.

Corynebacterium pseudotuberculosis is the cause of caseous lymphadenitis (CLA) in small ruminants, a chronic granulomatous disease that provokes significant zootechnics losses to ovine and goat breeders in northern Brazil. The present work was conducted to analyse aspects of humoral and cellular immune responses after experimental infection. Eight goats were infected intradermally with a single dose of virulent C. pseudotuberculosis strain and specific IgG, interferon-gamma (IFN-gamma) production as well as IgG avidity and antigens pattern recognition dynamics against an excreted-secreted antigen were recorded during 20 weeks. At the end of the follow-up, animals were slaughtered and necropsied. Although no animals showed apparent clinical signs of infection at the end of the trial, IFN-gamma response, even more so than the humoral response, differentiated animals into two groups of high or medium/low response. The time-course of IFN-gamma production presented a short-lived primary response on day 5 after infection of animals of both groups, and a strong and long lasting secondary response starting on day 16 after infection in the high response group. The indirect ELISA used was able to detect a positive antibody titre between 6 and 11 days after infection in the two groups. IgG avidity index oscillated initially between 15 and 45%, and showed approximately 5% units increment during the 20 follow-up weeks. With only one individual exception, the qualitative antigens pattern recognition showed on day 11 after infection remained constant through the experiment. IgG avidity is highly correlated with IgG production, but could not be related with specific immunodominant bands. Both humoral and cellular responses kinetics presented a similar pattern of activation/deactivation but necropsy results suggested that the IFN-gamma test would be a very specific marker of CLA status.

Isolation of Neospora caninum from the brain of a naturally infected dog, and production of encysted bradyzoites in gerbils.

Neospora caninum was isolated from the brain of an adult dog in Brazil. Cerebral tissue from the dog was inoculated into Mongolian gerbils. Gerbils were euthanized 3-4 months later and bradyzoite-containing tissue cysts were observed in their brains. N. caninum (designated NC-Bahia) was isolated in cell culture after inoculation with tissue cysts from the gerbils. The identity of the parasite was confirmed by immunohistochemical examination and polymerase chain reaction (PCR). Gerbils may be a useful alternative to immunosuppressed mice for isolation of N. caninum and for production of encysted bradyzoites.

Serum vascular endothelial growth factor in late rheumatoid arthritis.

OBJECTIVES: To investigate the serum levels of VEGF in patients with rheumatoid arthritis of long duration. METHODS: Serum VEGF levels were measured in 118 patients with long-standing rheumatoid arthritis according to the ACR criteria (mean duration 12 years). The disease activity score was evaluated by the method of van der Heijde et al. RESULTS: Serum levels of VEGF in patients with RA were significantly higher than in healthy controls. VEGF levels showed no correlation with CRP, SAA amyloid protein, or the disease activity score. CONCLUSIONS: Our findings suggest that, contrary to the results reported in patients with early onset RA, where VEGF appears to play an active part in joint inflammation, in long-standing RA elevated VEGF serum levels may be an independent marker although its significance remain to be established.

Modulation of the immune response by progesterone-induced lymphocyte factors.

Rat spleen and peripheral blood lymphocytes express progesterone receptors whose concentration is increased greatly during the early phase of pregnancy. After stimulation of progesterone the expression of receptors was augmented 2-3 times. When cells were cultured in the presence of progesterone they released a soluble factor that inhibited cellular immunoreactions (MLR, CRC) and cellular proliferation as measured by thymidine incorporation by spleen-cell culture. This factor also inhibited the synthesis of anti-DNP antibodies by a mouse hybridoma and diminished the proportion of cells in phase S. However, the percentage of asymmetric molecules produced by the hybridoma remained unaltered. These results support the hypothesis that soluble factors released by rat lymphocytes modulate the immune response of the mother and participate in the mechanism that protects the fetus against antipaternal antibodies.

Validation of models for predicting the use of health technologies.

Validation must be carried out before a model can be used confidently as a tool of managerial decision making in health care. The authors describe a bootstrap approach to validating models for predicting the utilization of four technologies used in neonatal care: measurement of blood gases (gasometry), the oxygen hood, continuous positive airway pressure (CPAP), and mechanical ventilation. These models were fitted by stepwise multiple linear regression from 20 prognostic covariates of 193 neonates. One hundred bootstrap samples were generated to validate the choices of covariates in the models based on their frequencies of selection. This approach validated the models for the oxygen hood and CPAP. The regression coefficients and standard deviations for the CPAP and oxygen hood models were estimated using 200 additional bootstrap samples. A close agreement between stepwise and bootstrap estimates was observed for both models. These results suggest that bootstrap can be useful for validating models for predicting the utilization of health technologies.

Sympathomimetic effects of Scoparia dulcis L. and catecholamines isolated from plant extracts.

The herb Scoparia dulcis L. is used in Brazilian folk medicine to treat bronchitis, gastric disorders, haemorrhoids, insect bites and skin wounds, and in oriental medicine to treat hypertension. A previous study has shown that extracts of S. dulcis have analgesic and anti-inflammatory properties; in this work the sympathomimetic activity of an ethanolic extract of Scoparia dulcis L. has been investigated in rodent preparations in-vivo and in-vitro. Administration of the extract (0.5-2 mg kg-1, i.v.) to anaesthetized rats produced dose-related hypertension blocked by the alpha-adrenoceptor antagonist prazosin (1 mg kg-1). Partition of the extract in chloroform-water yielded an aqueous phase 20 times more potent than the extract; this produced hypertension in either reserpine-treated or pithed rats. In untreated and reserpine-treated rats the same fraction (1-3 x 10(3) micrograms mL-1) produced concentration-dependent contractions of the vas deferens musculature parallel to those obtained with noradrenaline (10(-8)-10(-4)M). Prazosin (10(-7)M) reduced the maximum contractile effect of the aqueous fraction, and shifted the concentration-response curves for noradrenaline to the right. The aqueous fraction (25 and 50 micrograms mL-1) increased the inotropism of electrically driven left atria of rats, the effect being blocked by propranolol (0.4 microgram mL-1). In preparations of guinea-pig tracheal rings the aqueous fraction (1-3 x 10(3) micrograms mL-1) relaxed the muscle contraction induced by histamine (10(-4) M) in proportion to the concentration. The effect was antagonized competitively by propranolol (1.5 microM). High-performance liquid-chromatographic analysis of the aqueous fraction revealed the presence of both noradrenaline and adrenaline in the plant extract. The results indicated that both catecholamines may account for the hypertensive and inotropic effects obtained after parenteral administration of S. dulcis extracts. This sympathomimetic activity is, however, unrelated to the previously reported analgesic and anti-inflammatory properties of the plant extract, but may explain its effectiveness upon topical application in the healing of mucosal and skin wounds.

Influence of mouse placental factors on in vitro antibody synthesis.

Placental culture supernatants (PS) obtained from various mouse crossbreedings were added to mouse IgG1 hybridoma cultures producing anti-DNP antibodies. The quantity of monoclonal antibody (mAb) produced, the nature of these antibodies and the proliferation of the hybridoma cells were studied. It was observed that the supernatants increased or diminished the production of mAb, depending on the genetic origin of the placentae. This effect was the same using placentae from primiparous or multiparous females and it was not due to modifications of the cellular proliferation of the hybridoma, as shown by 3H-thymidine uptake. It was also found that placental supernatants induced an increase in the proportion of asymmetric, blocking antibodies synthesized by the hybridoma. This effect was detected with supernatants from both allogeneic or syngeneic crossbreedings, but only when placentae were obtained from multiparous females. These observations indicate that placentae produce at least two soluble factors that participate in the regulation of antibody synthesis and suggest that these factors play an important role in the immune equilibrium between mother and fetus.

Ornithine decarboxylase from Leishmania mexicana promastigotes: interaction with pyridoxal 5'-phosphate and alpha-difluoromethylornithine.

The catalytic properties of ornithine decarboxylase (ODC) from Leishmania mexicana as well as the interaction with its cofactor pyridoxal 5'-phosphate (PLP) and the irreversible inhibitor alpha-difluoromethylornithine (DFMO) have been studied using partially purified preparations of the enzyme obtained from parasite promastigotes. Leishmania extracts prepared in the presence of saturating concentrations of PLP yielded an enzyme considerably more resistant to heat inactivation and with a three-fold higher activity than the ODC obtained without the addition of cofactor. The complete removal of PLP by treatment with hydroxylamine yielded the apoenzyme which shows an absolute requirement for PLP to recover its enzymatic activity. The Km values for L-ornithine and PLP were 0.7 mM and 25 microM, respectively, while Ki for DFMO was 0.2 mM. The restoration of ODC activity from apoenzyme and cofactor seems to involve time and temperature-dependent activation processes. L. mexicana ODC has an apparent molecular mass of 240 +/- 20 kDa.